Abstract
High resolution fluorescence microscopy is a key tool in the elucidation of biological fine-structure, providing insights into the distribution and interactions of biomolecular systems down to the nanometer scale. Expansion microscopy is a recently developed approach to achieving nanoscale resolution in optical imaging. In the experiment, biological samples are embedded in a hydrogel, which is isotropicaly swollen. This physically pulls labels apart, allowing more of them to be resolved. However, in the gelation and swelling process, two factors combine to reduce the signal in the final image; signal dilution and the polymerization reaction, which can damage some fluorophores. Here, we show a chemical linking approach that allows covalent grafting of biomolecular target and reporter in expansion microscopy. Through the combination of a targeting ligand, a reporter moiety and a polymerizable group in a single linker, complex constructs can be prepared in a single, labelling step. We show application of this new series of molecules in the targeting of the cell cytoskeleton, a first example of lipid membranes in expansion microscopy; direct immunostaining with primary and secondary antibodies, and direct grafting of ISH probes and signal amplification initiators (HCR and RollFISH). Our probes allow direct, multiplexed targeting of the cellular blueprint and enable a range of novel imaging approaches in combination with expansion microscopy.
